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Funktionsanalyse von Androglobin, einem neuartigen, mit männlicher Infertilität assoziierten sauerstoffbindenden Protein

Fachliche Zuordnung Reproduktionsmedizin, Urologie
Anatomie und Physiologie
Förderung Förderung von 2016 bis 2020
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 317633050
 
Erstellungsjahr 2023

Zusammenfassung der Projektergebnisse

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans and is characterized as a chimeric protein with a calpain domain and a unique circularly permutated globin domain. This newest member of the globin family is evolutionary ancient and strongly conserved, being present in mammals, vertebrates, more basal animal clades and even unicellular organisms. We previously showed that Adgb expression is associated with postmeiotic stages of mammalian spermatogenesis. The present project aimed to further characterize expression, biochemical regulation and function of this novel globin type. On the protein level, we successfully expressed and purified the ADGB globin domain as well as full-length ADGB from baculovirally infected Spodoptera frugiperda (Sf9) insect cells. We found that the full protein context is necessary for a stably folded globin domain of androglobin. UV-Vis spectroscopy measurements indicated heme hexacoordination. Interestingly, we observed a time-dependent truncation of the full-length protein, possibly reflecting autocleavage, a phenomenon commonly observed among the Ca2+-dependent calpain proteases. On the mRNA level, we explored the cell-type-specific expression, regulation and function. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that -in addition to testes- ADGB is prominently expressed in the female reproductive tract, lungs and brain, specifically being associated with cell types forming motile cilia. This association was subsequently confirmed in a wide variety of other, evolutionarily basal model organisms such as cnidarians and sponges. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. We further investigated the ADGB gene regulatory landscape using various complementary approaches. We first characterized its promoter by mining epigenomic databases for accessible chromatin and promoter/enhancer-associated histone marks, and by performing exogenous promoter-dependent luciferase assays and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays further identified that FOXJ1, a crucial transcription factor of ciliogenesis, substantially enhances luciferase activity driven by the ADGB promoter. Accordingly, chromatin immunoprecipitation assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine-mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. Notably, under FOXJ1 overexpression conditions, endogenous ADGB mRNA levels were significantly increased. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. Furthermore, we illustrate a more complex transcriptional regulation of the ADGB locus by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicate an ADGB- dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB.

Projektbezogene Publikationen (Auswahl)

 
 

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